Structure and Function nACh receptor Flashcards
how can the different modes of the patch-clamp technique can be
exploited to study various aspects of receptor/ion channel function?
This technique allows resolution at real time single receptors going through the process of opening and closing.
A pipette was used to suck at high pressure, to form a high resistance seal (Gigahom seal) which electrically and mechanically tight.
Different paths can be taken; can put whole cell with open pore into salt solution and record receptor population
Can snap the receptor so its upside down and measure its activity by changing the concentration at the back.
Can pull electrode, pipette away from cell so it snaps off, giving access to an intracellular membrane.
What are the distinct gene families of transmitter-gated
ion channel subunits.
CYS-loop (pentamer)
Gluatmate (tetramer)
P2X (trimer)
What has cloning of receptor subunits led to?
The discovery of numerous receptor isoforms, which exhibit distinct
physiological properties showing they belong to the Cys-loop transmitter-gated ion channel family.
Multiple subunits means targets for developing novel therapeutics.
How fast can ions travel?
Ions flow selectively at a typical rate of 10,000,000 (10million) ions per second!!
What kind of receptor is nAChR?
CATION-CONDUCTING (non selective to positive substrates)
Cation non-selective channel: usually primarily conducting
Na+ Ca2+ in to the cell & K+ out of the cell.
What are the different modes of the patch- clamp technique?
1) Cell attached patch
2) Whole cell voltage clamp (removed)
3) Outside-out patch (reversed)
4) Inside- out patch (removed but not reversed)
What are the pros and cons of the Cell attached patch?
Advantages:
1) The intracellular milieu is maintained (no biochemical wash-out).
2) Control of the extracellular solution.
Disadvantages:
1) The agonist always present, ACh cannot be washed off and so receptor desensitization.
2) No control of the intracellular content.
What are the pros and cons of the Whole cell voltage patch?
Advantages:
1) Control of the intracellular & extracellular mileu.
2) Can record from a population of receptors/ion channels.
3) Can introduce biochemical modulators intracellularly via the pipette.
Disadvantages:
1) Biochemical “washout” of the intracellular mileu.
2) POOR Resolution- can rarely observe SINGLLE receptor activity
What are the pros and cons of the Outside-out patch?
Advantages:
1) Control of the intracellular & extracellular mileu.
2) Can change/control extracellular drug (agonist) concentrations.
Disadvantages:
1) Biochemical “washout” of the intracellular mileu.
2) Cannot change intracellular messengers eg. c-AMP
What are the pros and cons of the Inside-out patch?
Advantages:
1) Control of the intracellular & extracellular mileu.
2) Can introduce biochemical modulators eg. c-AMP to the intracellular face.
Disadvantages:
1) CANNOT change the concentration of agonist
Explain in what way the nAChR isopening is complex.
It binds to TWO ligands; when it is just bound o one ligand it only opens briefly, however when it binds to two it is open for longer.
Double binding makes opening more stable.
In nature, where were different nAChR found
In electric rays; electric organs need lots of ACh to discharge like a battery.
This gives evidence that similar subunits have developed in common ancestors.
Describe Cys-loop transmitter-gated ion channel family basic structure.
The family members are nAChR, GABAA, glycine & 5HT3 receptors.
All have a PENTAMERIC assembly of the subunits with binding sites found between the subunit interfaces.
Describe some of the different nAChR subunits?
2-9alpha, 2-4beta are neuronal
1alpha and 1beta are in muscle electric organ.
What are PAMs?
positive allosteric modulators that are new drugs being made (as well as receptors antagonists,
agonists)
PAM amplifies ACh effect (on their own with no ACh there is no effect)
Different subtypes means SPECIFIC BINDING.
What are some examples of disorders that have been linked to mutated receptors believed to be treated by drugs (PAM)?
Neuronal: Alzheimer’s Parkinson’s Schizophrenia Attention Deficit Disorder Anxiety Pain relief
What is a alpha7-5HT3A chimera?
It is a hybrid synthetically made using 5HT3areceptor and A7-nAChreceptor
What features does the chimera share with the original receptors?
It has the same agonist (ACh) as a7-nACh receptor (where 5-HT agonist is 5-HT)
It blocks a-bungarotoxin just like a7-nACh. (where 5-HT has no effect)
But it has a small channel conductance like 5-HT3areceptor
What is a ACh BP?
ACh binding protein binds the the ALPHA SUBUNIT and its adjacent subunit
It is the same size of the extra cellular domain of the whole receptor
Crystallography has shown that exists as a HOMOPENTAMER (lacking the transmembrane components)
It is found in snail brains where it mops up EXCESS ACh.
Describe the interaction between the AChBP and the agonist.
The agonist stabilises loop C of the protein in a contracted form, where open arms are shown to fold down so they no longer extend.
How do amino acids influence the receptor? What amino acids are important in selectively?
Extracellular and intracellular rings of negatively charged
glutamate and aspartate residues influence cation conductance.
Cationic nAChR made anionic by mutating Glu to Arg.
Anionic α1-GlyR to cationic by mutating Arg to Glu
Give an example of a variation seen in the 5-HT3 receptors. How does this compare with the nACh receptor?
Homomeric made up of the same subunit has a lower conductance
Heteromeric receptors have more than one type of subunits give a HIGHER CONDUCTANCE
However, homomeric nACh receptors have a HIGH conductance too
Through chimeria experiments using the process of elimination, what was found out about the receptor structure and conductance?
the large intracellular loop of an important determinant of single channel conductance.
It has been narrowed down to 21 amino acids on the loop
The receptor was Xrayed, what did this show?
There were 5 portals at the base for agonist exists like a watering can
